The overall goal of this project is to advance the proton-electron double-resonance technique, PEDRI, to a level allowing for functional mapping of physiologically-critical parameters, such as pH, oxygen, redox and intracellular glutathione (GSH), in animals and, potentially, in humans. To reach this goal we will use a new concept of Variable Radio Frequency (VRF) PEDRI in combination with an array of specific paramagnetic probes. This will allow for functional mapping within MRI high quality spatial resolution and short acquisition time. This new proposal is a logical extension of our recent R21 which was successful in validating the concept of functional PEDRI. The success of this proposal will open possibilities for bringing this new PEDRI technique into the leading research and clinical MRI centers. This proposal has promising potential taking into account that VRF PEDRI system allows for the simplified design by elimination of the field cycling coil assembly and its power supplies which will result in a complete utilization of the gap in the magnet system of conventional MRIs. The specific aims are: (SA1) To enable Variable Radio Frequency PEDRI for in vivo functional imaging. We will focus on the further development and optimization of VRF PEDRI, which is an instrumentally-innovative modification of PEDRI and has been found to be highly efficient for functional applications during the exploratory R21 phase. (SA2) To design PEDRI-oriented pH, oxygen, redox and GSH sensitive paramagnetic probes. The probes with functionally-dependent ratiometric spectral parameters, including dual function probes, with extended aqueous solubility and stability will be synthesized. Synthesis and optimization of the probes is absolutely essential for the overall efficiency of functional PEDRI applications. (SA3) To map in real-time the parameters of a tumor microenvironment using VRF PEDRI. The capacity of the VRF PEDRI for in vivo pH and oxygen mapping will be demonstrated for the most effective probes in PyMT transgenic mice that spontaneously-develop breast cancer. We plan to perform VRF PEDRI imaging and construct real-time, spatially-resolved and stage-specific signature profiles of tumor pO2, pH, redox, and GSH as the mammary tumors progress to malignancy. In summary, the success of this project may have a significant impact on the future of in vivo functional imaging applications to medicine.